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,
1
* Clinics of Physical Medicine and Rehabilitation and
Institute of Pathology, Medical Faculty (Charité), Humboldt University Berlin, D-10098 Berlin, Germany; and
Ethel Percy Andrus Gerontology Center, and Division of Molecular Biology, the University of Southern California, Los Angeles, California 90089-0191, USA
1Correspondence: Ethel Percy Andrus Gerontology Center, University of Southern California, 3715 McClintock Ave., Room 306, Los Angeles, CA 90089-0191, USA. E-mail: kelvin{at}usc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) of human BJ fibroblasts. During proliferative
senescence, we found a marked decline in all proteasome activities
(trypsin-like activity, chymotrypsin-like activity, and
peptidyl-glutamyl-hydrolyzing activity) and in lysosomal cathepsin
activity. Despite the loss of proteasome activity, there was no
concomitant change in cellular levels of actual proteasome protein
(immunoassays) or in the steady-state levels of mRNAs for essential
proteasome subunits. The decline in proteasome activities and lysosomal
cathepsin activities was accompanied by dramatic increases in the
accumulation of oxidized and cross-linked proteins. Furthermore, as
proliferation stage increased, cells exhibited a decreasing ability to
degrade the oxidatively damaged proteins generated by an acute,
experimentally applied oxidative stress. Thus, oxidized and
cross-linked proteins accumulated rapidly in cells of higher
proliferation stages. Our data are consistent with the hypothesis that
proteasome is progressively inhibited by small accumulations of
oxidized and cross-linked proteins during proliferative senescence
until late proliferation stages, when so much proteasome activity has
been lost that oxidized proteins accumulate at ever-increasing rates.
Lysosomes attempt to deal with the accumulating oxidized and
cross-linked proteins, but declining lysosomal cathepsin activity
apparently limits their effectiveness. This hypothesis, which may
explain the progressive intracellular accumulation of oxidized and
cross-linked proteins in aging, is further explored during postmitotic
senescence in the accompanying paper (1)
.—Sitte, N.,
Merker, K., von Zglinicki, T., Grune, T., Davies, K. J. A.
Protein oxidation and degradation during cellular senescence of human
BJ fibroblasts: part I—effects of proliferative senescence.
Key Words: aging • cross-linked proteins • lysosomes • protein oxidation • protein turnover • proteolysis • proteasome • senescence
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 3)
. Despite the great variety of
oxidative changes that can occur in the primary, secondary, and
tertiary structures of proteins, oxidatively modified proteins
generally seem to be selectively recognized and degraded by proteases
unless they first become extensively aggregated or cross-linked.
Partial refolding with exposure of (normally shielded) hydrophobic
residues plays a key role in the recognition of oxidized proteins
(4
, 5)
.
We (6
7
8)
and others (2
, 3
, 9
10
11)
have
reported that the proteolytic system responsible for the selective
degradation of oxidized proteins in mammalian cells is the proteasome.
Using selective proteasome inhibitors (8)
, proteasome
immunoprecipitation (6
, 7)
, or antisense oligonucleotides
against the proteasome C2 subunit to decrease the intracellular content
of proteasome, we were able to show that the selective degradation of
oxidized proteins in living cells requires proteasome (6
, 7)
. During the past several years many investigations have
reported the selective degradation of isolated oxidized model proteins
and of oxidatively modified intracellular proteins
(2
3
4
5
6
7
8
9
10
11
12
13
14)
. From such work it might be concluded that all
oxidized proteins undergo rapid and selective degradation; in fact, the
situation is much more complex. It was noticed early in our studies
that mildly oxidized proteins are the best substrates for proteolysis
whereas extensively oxidized proteins are actually resistant to
degradation and, instead, tend to aggregate and cross-link
(15)
. It has been shown that oxidatively modified,
aggregated, and cross-linked forms of proteins accumulate during normal
aging of cells (16
17
18
19
20
21)
, as well as in several
pathological conditions (16
, 22)
. Such observations have
been reported in organisms as diverse as flies, rats, and humans
(16
17
18
19
20
21)
.
The in vitro culture of normal human diploid fibroblasts has
long served as a model system for studying replicative senescence.
Human fibroblasts, like other nontransformed somatic cells, have a
finite replicative capacity (23
, 24)
; in other words,
these cells are mortal and undergo a limited number of population
doublings in culture, after which they enter a viable but
nonproliferative phase known as postmitotic senescence. Although it is
clear that this in vitro system is not the same as aging
in vivo, the model does offer several advantages for our
studies. First, the use of an in vitro senescence model
allowed us to differentiate between proliferative senescence (this
study) and the process of senescence in nondividing cells achieved by
cultivation of confluent cells under hyperoxic conditions (see ref
1
). In the present paper we used a Hayflick-like
senescence model of human fibroblasts to investigate cells of different
proliferation stages and test the influence of progressive cell
divisions on protein turnover, proteasome activity, lysosomal protease
activity, and the intracellular accumulation of oxidized proteins.
Using this model, complex biochemical investigations of living cells in
the same cell population were possible. Further advantages are that
there is no influence of extracellular matrix on standardization from
one experiment to another, and that the proliferative senescence (this
paper) and the senescence of nondividing cells (1)
could
be investigated in parallel. The accepted disadvantage of this model is
the lack of influence of other cell types and tissues during the
senescence process.
We undertook the present investigations with the goal of examining the
hypothesis that senescence is accompanied by changes in cellular
protein oxidation and protein turnover. Previous studies in tissues, in
cells isolated from young and old animals, and in vitro
(16
17
18
19
20
21
, 25
, 26)
have not revealed whether the
accumulation of oxidized proteins is a function of cells in the normal
mitotic cycle or of nondividing postmitotic cells. Previous studies
have investigated the effects of proliferative senescence on gene
expression (27)
, telomere shortening (28)
,
stress resistance (29)
, and turnover of total or
individual cell proteins (25
, 26)
in human fibroblasts,
and it seemed highly appropriate to use these cells for our studies.
The goal of our studies was to investigate for the first time in
parallel, the protein turnover of one cell line during the aging
process of proliferative senescence (the present paper) compared with
aging of nondividing cells 1
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oxidant treatment
Nearly confluent fibroblast cultures were treated for 30 min at
37°C with various concentrations of
H2O2 or paraquat
(5-methyl-viologen) at pH 7.4 in phosphate-buffered saline (PBS):
Control cultures were exposed to PBS alone. After this treatment, the
PBS was removed and the cells were cultured in normal medium for up to
48 h. For investigations with metabolically radiolabeled cellular
proteins, the radiolabeling procedure was performed prior to oxidant
treatment.
Measurement of overall proteolysis
The degradation of metabolically radiolabeled proteins in
confluent fibroblasts was measured after a 16 h labeling procedure
(6
, 7)
. During the labeling procedure, cells were
incubated with [35S]-methionine in
methionine-free minimal essential medium. After 16 h of incubation
at 37°C, the nonincorporated label was removed and the cells were
washed twice with PBS. Cells were next treated with
H2O2 or paraquat, or used
as controls, as described above. The degradation of metabolically
radiolabeled proteins was quantified, after addition of an equal volume
of 20% trichloroacetic acid, by measuring (liquid scintillation
counter) the production of acid-soluble counts in the supernatant after
centrifugation at 14,000 g for 10 min.
Measurement of proteolytic activities
The maximal activities of lysosomal cathepsins were analyzed
according Inubushi et al. (30)
, whereas proteasome
activity was determined as described by Grune et al. (6)
.
Between 0.3 and 1.6 x 106 cells were washed
twice with PBS and lysed in 150 µl of water containing 1.0 mM
dithiothreitol during vigorous shaking for 1 h at 4°C. The
lysates were immediately used to determine proteolytic activities.
Proteasome activity
The remaining unlysed cells, membranes, and nuclei were removed
by centrifugation at 14,000 g for 30 min. The supernatant
was incubated in a buffer consisting of 50 mM Tris-HCl (pH 7.8), 20 mM
KCl, 0.5 mM Mg-acetate, and 1 mM dithiothreitol. The fluoropeptide
substrate suc-LLVY-MCA was used to measure the chymotrypsin-like
activity of the proteasome; z-PFR-MCA was used for the trypsin-like
activity and z-LLE-ßNA for the peptidyl-glutamyl-hydrolyzing
activity. After a 1 h incubation with 200 µM of one of these
fluorogenic peptides, hydrolysis was stopped by addition of an equal
volume of ice-cold ethanol and by further dilution with 0.125 M sodium
borate (pH 9.0). The fluorescence of the reaction products was
monitored at 380 nm excitation and 440 nm emission for MCA, and at 335
nm excitation and 410 nm emission for ßNA using free MCA or ßNA,
respectively, as standards.
Activity of lysosomal cathepsins
Lysates were sonicated for 2 min on ice in a SONOPLUS GM70. The
proteolytic activity assay was performed by incubation of lysates at
37°C for 30 min in a buffer containing 50 mM sodium acetate (pH 5.5),
8 mM cysteine hydrochloride, and 1 mM EDTA in the presence of 200 µM
z-FR-MCA as a fluorogenic peptide substrate. The reaction was
terminated by addition of an equal volume of ice-cold ethanol;
measurements of MCA release were performed as described for the
determination of proteasome activity above.
Protein carbonyl measurement
Protein carbonyl content was determined in cell lysates (4
mg/ml) by the ELISA of Buss et al. (31)
with modifications
by Sitte et al. (8)
. The detection system employed an
anti-dinitrophenyl rabbit immunoglobulin G (IgG) antiserum (Sigma,
Deisenhofen, Germany) as primary antibody and a monoclonal anti-rabbit
IgG antibody peroxidase conjugate (Sigma) as secondary antibody.
Development was achieved with o-phenylenediamine.
Malonyldialdehyde measurement
Determination of the lipid peroxidation product
malonyldialdehyde (MDA) was performed according to the method of Wong
et al. (32)
with modifications by Sommerburg et al.
(33)
. Cell pellets were boiled briefly in the presence of
thiobarbituric acid for 60 min. The reaction was stopped by cooling the
samples in an ice bath. The neutralized samples were analyzed on an
isocratic reversed-phase-HPLC system using a Supelcosil column
(Supelco, Deisenhofen, Germany; 150x4 mm LC-18-S; 5 µM) and a
potassium phosphate buffer/methanol eluent. Detection was performed by
fluorescence (excitation, 525 nm; emission, 550 nm).
Oxidized and cross-linked proteins
Oxidized/cross-linked proteins (lipofuscin-like or ceroid-like
material) in samples of 
3 x 105
cells were determined by measuring the cellular autofluorescence in the
yellow-green range of the spectrum (563–607 nm) by flow cytometry
using a BECTON-DICKINSON FACScan, as described previously
(34)
.
Immunoblots
After equalizing the protein content of centrifuged cell lysates
(14,000 g for 30 min) sodium dodecylsulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) was performed according to Laemmli et
al. (35)
under reducing conditions. Proteins were
transferred from SDS-PAGE gels to nitrocellulose membranes using a
Bio-Rad Trans-Blot apparatus and incubated with a polyclonal
anti-rabbit proteasome antibody (Affinity, Exeter, U.K.). The secondary
antibody was an anti-rabbit IgG peroxidase conjugate, which was
detected by chemiluminescence using the ECL assay (Amersham, Little
Chalfont, U.K.).
Northern blots
RNA was extracted from BJ fibroblasts using ‘RNeasy Mini kits’
(Qiagen, Hilden, Germany). Samples containing 15 µg of RNA were
loaded on a 1.2% agarose gel. Northern blots were performed by
conventional procedures as described by Nakamura et al.
(36)
. cDNA probes containing the proteasome C9-(
)
subunit and the N3-(ß) subunit genes were a kind gift of Dr. U.
Kuckelkorn and Prof. P.-M. Kloetzel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For BJ cells—a human
foreskin fibroblast cell line—a total of 85–90 PDs has been
reported (37)
. We decided to use this cell line because we
could cultivate these cells for almost 45 proliferation
doublings(Fig. 1A
). This means with our experiments we were able to cover
50% of the total proliferative potential of this cell line. We used
cell cultures at different proliferation stages to investigate
proliferative senescence. Since cells during proliferative senescence
change their morphological features dramatically we demonstrate in Fig. 1C
the changes in shape, size, and appearance of young and
old cells.
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Protein synthesis and protein degradation
As shown in Fig. 1B
, protein synthesis exhibits a
moderate decline during proliferative senescence. Next we were
interested in protein degradation during proliferative senescence, both
the activities of lysosomal proteases and the activity of the
proteasome. Degradation of the fluorogenic peptide z-FR-MCA in cell
lysates under acidic conditions gives a good approximation of the
activity of lysosomal cathepsins (30)
, whereas in
centrifuged (membrane-free) lysates of cells the proteolytic activity
of the proteasome is detectable under alkaline conditions by hydrolysis
of three other fluorogenic peptide substrates, each specific for one of
the main hydrolyzing activities of this protease. As reported in
Fig. 2A
, B
, a decrease in both lysosomal protease activity and all
three cytosolic proteasome activities occurs during proliferative
aging. Both the lysosomal and the proteasomal proteolytic systems lose
from 60–80% of their initial activities, indicating a dramatic loss
in the overall capacity of cells to degrade proteins. Although all
proteasome activities decline significantly, the
peptidyl-glutamyl-hydrolyzing activity declines to almost zero during
proliferative senescence (Fig. 2A
)
|
Since it is known that the proteasome is able to recognize and
selectively degrade oxidized proteins in isolated systems, we wanted to
test the activity of this enzyme complex in young and old cells during
the degradation of standardized oxidized proteins (Fig. 3
). For these studies, we incubated cell lysates from BJ fibroblasts with
PDs of 46 or 74 with the standardized,
H2O2 oxidized proteolysis
substrate [3H]myoglobin. The results of Fig. 3
indicate that old cells are not able to degrade the oxidized protein
substrate to the same extent as can young cells.
|
The results of Fig. 2A
and Fig. 3
indicate that proteasome
activity declines during proliferative senescence and that the
proteasome-dependent ability to degrade oxidatively damaged proteins
undergoes a similar decline. To investigate whether this decline is due
to a decreased specific activity of the proteasome or to a decreased
total amount of the enzyme complex, we investigated cellular proteasome
content by immunoblots. As shown in Fig. 2C
, we found no
decline in proteasome band intensity, indicating that the proteasome
content of cells does not decline significantly. Northern blot analysis
(Fig. 2C
) also showed no variation in the levels of mRNA for
the C9-() and the N3-(ß) subunits of the proteasome. We therefore
conclude that the loss of proteasome activity during proliferative
senescence seems to be more an effect of enzyme inhibition or altered
regulation than a decreased amount of proteasome.
Accumulation of oxidized proteins during proliferative senescence
We hypothesized that loss of proteasome function might be
caused by an accumulation of oxidized proteins within aging cells. To
test the feasibility of this hypothesis, we first investigated several
parameters of protein/cellular oxidation during proliferative
senescence. As demonstrated in Fig. 4A
, a continuous increase in protein-bound carbonyl moieties,
an established marker of protein oxidation, was observed during
proliferative aging of BJ fibroblasts. Undegraded oxidized proteins
tend to cross-link and form highly polymerized protein aggregates
(15
, 38)
. Figure 4B
shows a distinct increase
of MDA during proliferative senescence. The role of MDA during the
formation of cross-linked aggregates is known, and protein aggregates
seem to combine with several of these oxidized cellular materials to
form the so called age-pigments, such as lipofuscin or ceroid-like
material (22)
. Since oxidized/cross-linked proteins have
an autofluorescence, we measured the appearance of cellular
autofluorescence by flow cytometry. As reported in Fig. 4C
, we discovered a progressive increase in cellular autofluorescence
during proliferative senescence.
|
Decline in normal and stress-induced overall proteolysis
To further explore our hypothesis that loss of proteasome function
might be caused by an accumulation of oxidized proteins as cells
approach proliferative senescence, we measured the overall turnover of
metabolically radiolabeled proteins in human BJ fibroblasts at
different proliferation stages. As demonstrated in Fig. 5A
, B
, a severe decline in overall proteolysis in response to
the oxidative stress of either
H2O2 or paraquat, was
measured during proliferative aging. Therefore, it would appear that
cells at late proliferation stages have a severely diminished ability
to appropriately remove oxidized proteins under conditions of oxidative
stress. As we have previously reported, mild oxidative stress generates
excellent proteolytic substrates that are rapidly degraded by the
proteasome in a process that prevents cellular accumulation (5
, 6
, 15)
. In contrast, more severely oxidized proteins exhibit
increasing aggregation and cross-linking, and become progressively
poorer substrates for the proteasome (5
, 6
, 11
, 12
, 15
, 38)
. With this background in mind, note that proliferative
senescence also generally decreased the oxidant concentration at which
maximal proteolysis occurred (Fig. 5A
, B
),
presumably resulting in increased accumulation of oxidatively modified
proteins at lower oxidant exposures.
|
Enhanced stress-induced accumulation of oxidized proteins in
senescent cells
Although we hypothesized that loss of proteasome function might be
caused by an accumulation of oxidized proteins within senescing cells,
the diminished proteolytic response of older cells to oxidants seen in
Fig. 3
and Fig. 5
might also be explained by diminished production of
oxidized proteins during acute oxidative stress. When we measured the
actual production of oxidized proteins in cells exposed to hydrogen
peroxide, we found that the peroxide-induced accumulation of
oxidatively modified proteins increased with population doublings
(Fig. 6
). Even when one subtracts the increased background level of
protein-bound carbonyls in untreated older cells, we still observed up
to a threefold increase in carbonyl levels in peroxide-treated
near-senescent cells (Fig. 6)
. Important to our hypothesis of
proteasome dysfunction in aging, Fig. 6
also reveals that cells at a PD
of 74 failed to degrade much of their accumulated protein-bound
carbonyl material even 24 h after peroxide exposure, whereas cells
at a PD of 46 had removed most of the carbonyl-containing proteins
within this time period.
|
We next examined the resistance of the proteasome and lysosomal
proteases after hydrogen peroxide treatment. As demonstrated in
Fig. 7
, there were no significant changes in the proteolytic behavior of these
proteolytic systems after treatment with moderate concentrations of
oxidant. In contrast, both the proteasome and lysosomal proteolysis
declined after exposure to 1.0 mM
H2O2 and although young
cells recovered after 24 h, the oldest cells showed no recovery of
activity (Fig. 7)
, indicating a decreased resilience of proteolytic
systems to oxidative inactivation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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17
18
19
20
21)
. Several authors have
speculated that this is the result of ineffective removal of oxidized
proteins, caused by an age-dependent decline in the proteolytic
machinery responsible for their degradation (15
, 18
, 38
39
40
41)
. In fact, Carney et al. (20)
, Stadtman et
al. (38)
, Starke-Reed and Oliver (39)
, and
Agarwal and Sohal (41)
have all reported declining neutral
alkaline protease activity (mostly proteasome) in brain and liver
during aging. These experiments were performed in whole tissues, which
of course are comprised of cells in various phases of the cell cycle
and at various PD levels. Therefore, we decided to investigate
senescence-related proteasome activity during both proliferative
senescence and the senescence of nondividing cells (1)
,
for the first time.
Beginning with the studies of Hayflick (23)
, many
researchers have studied changes in cellular metabolism during the
proliferation of primary cell cultures. The influence of
‘mis-translation’ on the proteolytic susceptibility of proteins in
senescent cells was revealed by Schimke and Hayflick (25
, 26)
, who demonstrated declining protein turnover in late phase
III fibroblasts. Since these cells are essentially postmitotic we
wondered whether decreases in protein turnover might actually occur
gradually during fibroblast proliferation. Therefore, we investigated
the activities of both the proteasomal and the lysosomal systems during
proliferation of BJ fibroblasts, and found a dramatic decline in the
activity of both systems.
The activity of the proteasome toward various fluorogenic peptides
declined proportionally, suggesting some overall mechanism of
proteasome inactivation rather than a site-specific inhibition of any
particular proteolytic site. We focused our further studies on the
investigation of the proteasomal system, since the proteasome seems to
be largely responsible for the degradation of oxidized cellular
proteins (2
3
4
5
6
7
8
9
10
11
12)
. Since no ‘age-related’ changes in
cellular proteasome content were observed by (polyclonal) immuno-blots
for the enzyme complex and since Northern blots for two proteasome
subunit mRNAs were also unchanged, it appears that the proteasome must
be down-regulated/inhibited/inactivated on a post-translational level.
Since it is known from the literature that the proteasome is
inhibited by oxidized proteins and cross-linked proteins (5
, 11
, 12
, 40)
, we tested for the presence of protein oxidation
products and, indeed, found that proliferative senescence is
accompanied by increased accumulation of oxidized proteins, as
demonstrated by protein carbonyl measurements and by the measurement of
cross-linked fluorescent material. The accumulation of lipofuscin or
ceroid-like fluorescent material has long been known as an
age-associated process (22)
, and so we decided to
investigate whether the accumulation of oxidized proteins in our model
is actually a consequence of oxidative stress. Using various
nonsenescent permanent cell lines, we have previously demonstrated that
oxidative stress is accompanied by enhanced protein turnover (6
, 7)
. This increase in protein turnover is strongly dependent on
the activity of the proteasome, as revealed by immunoprecipitation and
antisense experiments (6
, 7)
, and causes the removal of
oxidized proteins (8)
. We now report that senescing BJ
fibroblasts cells gradually lose the capacity for increased proteolysis
in response to the oxidative stresses of hydrogen peroxide or paraquat.
In the same experiments, cells exposed to oxidative stress exhibit a
progressive accumulation of protein oxidation products with increasing
senescence. Only very high concentrations of
H2O2 or paraquat directly
inactivated proteasomal or lysosomal activities in BJ fibroblasts, as
previously reported for nonsenescent cells (42
, 43)
,
ruling out direct oxidative inactivation of proteolysis as the cause
for subsequent accumulation of oxidized proteins.
Our studies reveal a clear decline in proteasome activity during
proliferative senescence and a diminishing responsiveness of the
proteasome to acute oxidative stress. These processes are accompanied
by an increased accumulation of oxidized proteins. We propose that
these processes are interactive and mutually propagating. In other
words, we suggest that a constant minor accumulation of a small number
of oxidized/cross-linked protein molecules occurs throughout life,
because some oxidized proteins will always ‘escape’ the proteasome.
Eventually the cellular concentrations of these accumulating protein
oxidation products reach a level that causes a generalized inhibition
of the proteasome, because they bind but cannot be degraded. The
consequent decrease in effective cellular proteasome activity causes a
more rapidly diminishing ability to degrade oxidized proteins;
therefore, accumulation of protein oxidation products occurs more
rapidly during the latter stages of proliferative senescence. In the
accompanying paper (1)
we have tested these ideas in
postmitotic and nondividing BJ fibroblasts.
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ACKNOWLEDGMENTS |
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Received for publication March 28, 2000. Accepted for publication June 6, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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